To evaluate the newly developed next generation sequencing assay by analyzing patients with isolated eye abnormalities collected in earlier studies including patients enrolled through eyeGENE program: This project started several years ago in the OGFVB/NEI. We developed a screening tool by the resequencing CHIP on 93 genes provided an opportunity for these samples from patients enrolled in NEI clinical research for years. Our manuscript was published in the IVOS later in 2011. We lately adapted next generation sequencing (NGS) technology. We developed a new panel composed of 184 genes related to retinal function and development. By NGS and microdroplet PCR technology, we have been more actively analyzing patient with variety categories of retinal dystrophies in a more efficient and accurate approach. We hired a company, RainDance Technologies, Inc., in designing a primer library for Retinal Dystrophy Panel (RD panel). Using the developed primer library, RainDance Technologies, Inc, we have performed NGS sequencing for more than 150 patients with variety of retinal diseases. Again, Dr. Fanns group from ITBP/NINDS helped the data processing and bioinfomatic analysis. We have identified more than 100 candidate mutations. With the efforts of lab technicians, summer interns and special volunteers, we have been working on verification of the identified targets by Sanger sequencing. Molecular mechanisms in many patients with sporadic retinal diseases or differential diagnostic difficulties have been revealed by this approach. Several cases went through the clinical verification by an outside CLIA lab and have been reported back to clinicians/patients. We are summarizing the data and preparing for manuscript. We are continuing the procedures to analyze more patients with RP. Those samples have been sequenced in our newly purchased MiSeq next generation Sequencer from Illumina. The data is now in processing and bioinformatic analysis at ITBP/NINDS. To develop new screening tool for corneal disease related genes: We continued our collaboration with the RainDance Technologies, Inc., in designing another primer library for Corneal Dystrophy Panel (CD panel) covering 60 genes. Using the developed primer library, we have analyzed 24 samples from patients with variety of corneal dystrophies. The data has been processed at the ITBP/NINDS. Mutations in known genes responsible for corneal dystrophies have been identified and verification by Sanger sequencing is in progress. To develop genetic tests for more OCA genes as collaboration with NHGRI for the Clinical, Cellular and Molecular Investigations into Oculocutaneous Albinism project: We have been established a close collaboration with Dr. David Adams from NHGRI for the Clinical, Cellular and Molecular Investigations into Oculocutaneous Albinism project. This collaboration includes development of research gene screening tests and validation of clinical genetic testing for the research project. We have developed OCA2 gene research test and later clinically validated the test. We have studied 40 patients and family members for the project. We recently had a publication in the journal of Human Mutations for a summary of this collaboration. To study GPR143 for Ocular Albinism (OA1) To expand our capacity to study the patients with albinisms, we have developed an assay to sequencing coding regions of OA1 (GPR143) gene for mutation screening. We are working on patients without mutations in other four OCA genes. To analyze the PAX2 gene structure in patients collected in earlier studies: This is a continuation of my clinical ophthalmic research from Minnesota and collaboration. We developed a clinical DNA testing of PAX2 gene for Renal-coloboma Syndrome and have been collecting tested samples for genetic heterogeneity analysis. After I joined NEI, we are extending this study. We have collaborated with Dr. Brain Brooks to study more patients in their PAX2 genes. We are extending the study with a hope that we could validate this protocol to a clinical test. We are planning to extend the study by screening exome of patients for new genes. To analyze promoter and intronic genomic abnormalities in Tyrosinase gene: This is also a continuation of my clinical research from Minnesota. I have been collecting tested samples for genetic heterogeneity analysis of TRY in Ocular-Cutaneous Albinism. Our research fellow, who joined my lab two years ago and unfortunately left last year because of family reasons, had been working on this project and made progress. She performed analysis on the correlation of the methylation with regulation of gene expression at mRNA level through several cell lines and developing different approaches to identify enhancer/suppressor regions. We have been analyzing the data remotely. It has been a difficulty with no refilling of the fellow position for research projects in DDL. However, we managed to deal with the difficulties and keep the analysis ongoing. We submitted an abstract to the ASHG 2013 annual conference. The abstract has been accepted. We are preparing the poster and I will present the poster upon the approval for traveling. To evaluate retinal related genes for mutation in patients with Occult Macular Syndrome for genetic modification: Occult Macular Dystrophy is a very unique macular dystrophy. Clinical diagnosis is usually difficult and fully based on multifocal ERG. Recently, such disorder was found related to mutations in a gene called RP1L1 in Japanese patients and verified in other ethnic populations. NEI ophthalmologists have been clinically evaluating patients with OMD and we have been developing a research procedure to screening the entire coding region of RP1L1 gene. With several students efforts, we found the reported mutations in 3 of 6 index patients. However, published patient analysis indicated a reduced penetrance of OMD in individual carrying the mutation. We are continuing the analysis to find if there is a genetic modification of such reduction. We are planning to publish the study once we finish the segregation analysis. To develop RNA sequencing based assay to generate transcriptome profiling in different cell types including the cell obtained through procedures modified from iPS protocols: By our NGS project, we have screening patients for mutations in retinal related genes. We have found known mutations in the known genes for the clinical conditions; we have found novel variations predicted as or likely mutations in the known genes for the clinical conditions; and we found known mutations not previously correlated to the clinical conditions. For those known mutations or predicted mutations in known genes, we can interpret by the evidences for the predicted functional consequences. However, we frequently found variations with uncertainty for their functional consequences in those known genes and we occasionally found known mutations in irrelevant genes for other retinal conditions in patients with retinal dystrophies. To clarify the correlation or exclude the functional involvement, we need to study the functional consequences of candidate variations at an alternative level such as but not limited to transcription and/or translation. As many retinal genes do not express in PBMC, we could study gene exress directly in white blood cells. Therefore, we are looking into evaluation of newly identified variation in a personal model system, such as skin biopsy or induced pluripotent stem cells. By collaboration with Dr. David Adams from NHGRI and Dr. Kapil Bharti from our own Branch at NEI/OGVFB, we are adapting the technology and develop the assays to study the functional consequences of variations.